Natural products have been a prolific source of therapeutics for combatting cancers, including the widely-used breast cancer drug taxol produced by the bark of the Pacific yew that acts through targeting tubulin to impair breast cancer pathogenicity. While there are countless natural products derived from plants, microbes, and other living organisms that have been shown to exert anti-cancer activity, the mechanism of action of most of these natural products remain poorly understood. One such natural product is nimbolide, a triterpenoid obtained from Azadirachta indica or neem, that has been shown by many groups to exert anti-cancer activity against multiple different types of cancers, including breast cancers, hepatocellular carcinomas, colon cancers, and renal cell carcinomas. While this natural product possesses compelling anti-cancer properties, the direct targets remain poorly understood. I have used an innovative chemoproteomic platform termed activity- based protein profiling (ABPP), which uses reactivity-based chemical probes to map reactive, functional, and druggable hotspots in complex proteomes, to map the proteome-wide ligandable hotspots targeted by the anti-cancer natural product nimbolide in breast cancer cells. My preliminary data using ABPP indicate that nimbolide selectively targets C8 on the E3 ubiquitin ligase RNF114 in 231MFP triple-negative breast cancer (TNBC) cells, leading to impaired ubiquitination of the tumor suppressor p21 through an impairment in the ability of RNF114 to recognize its protein substrates. This in-turn leads to an elevation in p21 levels, leading to impaired breast cancer cell pathogenicity. In this proposal, I will use innovative chemical biology approaches to determine the anti-cancer mechanisms of nimbolide and characterize RNF114 as a target for cancer therapy and for targeted protein degradation applications.